Pharmaceutical composition having a cationic excipient

ABSTRACT

The invention relates to a pharmaceutical composition comprising a pharmaceutically effective amount of a pharmaceutically active substance and a pharmaceutically acceptable carrier comprising a cationic excipient, wherein said cationic excipient is a labile ester of betaine and a lipophilic alcohol having at least one primary hydroxyl group. The invention also relates to the use of a labile ester of betaine and a lipophilic alcohol having at least a primary hydroxyl group as a cationic excipient in a carrier for a pharmaceutical composition comprising a pharmaceutically active substance.

TECHNICAL FIELD

The present invention relates to the field of pharmaceuticalcompositions comprising a cationic excipient as a carrier ingredient.More specifically, the invention relates to a new group of cationicexcipients for such compositions.

BACKGROUND OF THE INVENTION

Positively charged molecules have over the years been evaluated ascomponents of various types of drug delivery systems. Electrostaticinteractions with the drug molecule, a component in the biologicalsystem or in some cases both are often essential for the mode of actionof these types of excipients.

How fast a drug enters the systemic circulation after administrationdepends on several factors like the chemical characteristics of thesubstance (e.g. solubility, membrane permeability), the route ofadministration and the composition of the formulation. Selection ofexcipients for formulation of biological active materials like drugsmust focus on therapeutic efficiency of the dosage form but thecomposition of the formulation can also affect other important factorslike toxicity, drug load, storage stability and production costs.

There are several examples in the literature of cationic drug deliverysystems containing lipids. It has for instance been suggested that bypromoting nonbilayer structures in the cell membrane, lipids facilitatethe intracellular delivery of macromolecules. Encapsulation in cationicliposomes has been shown to protect proteins and peptides againstdegradation by enzymes in biological fluids. Cationic lipid containingsystems like emulsions and microemulsions have also been used to improvebioavailability after oral administration of sparingly water solubledrugs.

The development of novel drug formulations is often limited by the lackof safe and reliable excipients. A major problem with the use ofsurfactants in drug delivery systems is the potential toxicity ofsurface active agents. A balance between the efficacy in a carriersystem for drugs and the toxic effects is always of major concern forsubstances involved in drug delivery. We have now found that a class oflabile cationic substances, earlier demonstrated to have antimicrobialactivity, have unique potential to be used as cationic excipients fordrug delivery. The betaine ester, viz. betaine esterified with alipophilic alcohol, is a cationic surfactant with outstanding drugdelivery properties. Furthermore, the labile bond of the betaine esterresults in a cationic hydrolysis product, betaine, which is a normalhuman metabolite. This means that the toxicity related to the cationicsurfactant is transient.

The surface active betaine esters used in the present invention cantogether with drugs, and optionally other excipients, form aggregateslike micelles, microemulsions, emulsions, dispersions and liquidcrystalline phases in presence of water or biological fluids. Sincebetaine esters form complex with negatively charged polymers, likemucin, they are anticipated to retain the solubilized or dispersed drugsclose to the absorption site without damaging the tissue. This is afeature that is especially interesting for transmucosal drug delivery.Transmucosal delivery at sites where enzymatic degradation can occur orwhich has a pH of 6.0 or higher should be of particular interest fordrug delivery systems containing excipients with this type of labileesters. Drug delivery systems of negatively charged or sparingly watersoluble drugs are also of special interest for this invention.

The use of a betaine ester as part of a carrier system forpharmaceutical applications is disclosed e.g. in U.S. Pat. No. 5,492,937which describes a carrier composition which is a liquid at or below roomtemperature and forms a high viscosity layer or gel at body temperature,characterized in comprising a water-soluble, nonionic cellulose etherhaving a cloud point not higher than 40° C., a charged surfactant andoptional additives in water, wherein said optional additives areselected from the group consisting of flavouring agents, colorants,preservatives, isotonic agents and mixtures thereof, and in that thecombined concentration of the water-soluble, nonionic cellulose etherand the surfactant is below 3% by weight, and wherein the remainder ofthe composition is water and said optional additives. The origin of thegel formation is a strong hydrophobic interaction between polymer andsurfactant, which is cooperative in nature and thus resembles normalmicelle formation. Surfactant clusters formed in this way may then actas cross-links between different polymer chains, giving rise to anextended three-dimensional gel structure. The surfactant should containeither a positively or a negatively charged headgroup. Examples of theformer surfactants are alkyl ammonium compounds (e.g.hexadecyltrimethylammonium, tetradecylbetainate and hexadecylpyridiniumsalts, e.g. chloride and bromide). Thus, said carrier composition isaqueous and does not work the same way as the present invention.

U.S. Pat. No. 6,007,826 discloses a pharmaceutical or cosmeticcomposition comprising a pharmaceutically or cosmetically activeeffective amount of a hydrophobic active ingredient and a carrier, thecarrier being an oil-in-water type emulsion which comprises colloidparticles having an oily core surrounded by an interfacial film, saidactive ingredient being incorporated into said oily core, wherein saidinterfacial film comprises a combination of three different types ofsurface active compounds, a cationic lipid, a nonionic surfactant and ananionic surfactant or anionic lipid. Said cationic lipid is present in aconcentration of 0.05-2% by weight and is selected from the groupconsisting of a C10-C24 primary alkylamine, a C10-C24 primaryalkanolamine and a cholesterol ester (e.g. cholosteryl betainate).Cholesteryl betainate, a molecule with a large rigid steroid carbon ringstructured with an esterified secondary alcohol has, however, propertiesquite different from those of the betainates used according to thepresent invention.

SUMMARY OF THE INVENTION

The object of the invention is to provide a pharmaceutical compositioncomprising a pharmacologically active substance, generally a drug, and acarrier therefor comprising a new, specific type of cationic excipientwhich imparts good delivery or release characteristics to saidcomposition.

Another object of the invention is to provide a composition theexcipient of which is labile in the presence of water or aqueous bodyfluid so as to degrade into non-toxic products in the recipient body,generally a human being.

Still another object is to provide the use of said specific type ofcationic excipient in a carrier for a pharmaceutical composition.

Other objects of the invention should be apparent to the reader of themore detailed disclosure of the invention presented herein.

The objects of the invention are achieved by a pharmaceuticalcomposition as claimed in claim 1 and by the use as claimed in theindependent use claims.

Preferable embodiments of the composition as well as the use referred toare as claimed in sub-ordinated claims or specifically disclosed in thespecification below.

More specifically this means that the cationic excipient of the presentinvention is a labile ester of betaine and a lipophilic alcohol havingat least one primary hydroxyl group.

It is preferred that the carrier or composition is substantiallynon-aqueous.

Said labile ester has been shown to work very well as a carrier for drugdelivery and by said non-aqueous state of the carrier or composition the“lability” does not lead to any hydrolysis of the ester until in theanimal (mammal), generally human, body. When degraded the ester willthen result in the hydrolysis product betaine, which is a normal humanmetabolite. Thus, the toxicity generally related to the cationicsurfactant can be said to be transient in connection with the presentinvention.

By “labile” in connection with the present invention is generally meantan ester which undergoes hydrolysis to more than 50% during 24 h at pH7.4 in the presence of water or other aqueous liquid.

The term “substantially non-aqueous” generally means that such acondition does not cause any substantial hydrolysis of said labileester, e.g. less than 10%, or less than 5%, in the composition referredto.

Another definition of “substantially non-aqueous” is that generally atmost 5% by weight, preferably at most 2% by weight, most preferably atmost 1% by weight, of water is present in the composition. A similarterm having a similar meaning would be substantially water-free.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows results from Example 2 below.

FIG. 2 shows results from Example 4 below.

DESCRIPTION OF PREFERABLE EMBODIMENTS

The labile betaine ester used according to the present invention ispreferably an ester of betaine and an alcohol of the formulaR—CH₂—OH.

Thus, firstly a primary alcohol is used.

Secondly, R is a saturated or unsaturated aliphatic hydrocarbon residue,said unsaturation generally including double bonds only.

The carbon atom number of said symbol R is preferably 7-30, morepreferably 7-22.

This in turn means that the cationic betaine esters referred to willhave the formula:R—CH₂—OCO—CH₂—N (CH₃)₂ ⁺X⁻where X⁻ is a suitable counterion which is selected in accordance withknown principles per se for surface-active cationic surfactants.

A suitable counterion may be chloride or bromide.

Like for all ionic surfactants the physico-chemical properties of abetaine ester surfactant are mainly governed by the length of thehydrophobic tail of the molecule, i.e. by the R residue in the aboveformula. The water solubility and the critical micelle concentration(CMC) of the betaine ester both decrease with increasing numbers ofcarbon atoms of R.

Versatile esters are esters wherein R has 9-13 carbon atoms.

As said, the alcohol referred to is a primary alcohol. It may, however,well also contain further hydroxyl groups, primary as well as secondaryhydroxyl groups. That is, R is not necessarily an unsubstituted residue,but can be substituted, provided that the objects of the invention willnot be lost. Expressed in another way, the substitution may even improvethe characteristics of the cationic excipient, or composition, by theincorporation of substituents on R. Preferred substituents are primaryand/or secondary hydroxyl groups, preferably one or two of each thereof.

Other substituents may be selected in accordance with known principlesfor cationic surfactants.

The hydrocarbon residue R need not either be a chain with carbon atomsonly, i.e. a pure alkyl or alkenyl chain, but may well be interrupted byheteroatoms, such as O and/or N. According to a preferable embodiment ofthe invention at least one, preferably one or two, oxygen atom(s) is(are) present.

Preferably such oxygen atom(s) is (are) present in or as ester linkages.

More preferably said ester linkages are glyceride linkages, especiallypreferable examples being a 1-monoglyceride or a 2-monoglyceride.

Alternatively, the hydrocarbon residue R may comprise more than onetail, as exemplified by a diglyceride of the formula

Alternatively, the hydrofobic moiety of the betaine ester may comprisemore than one head group, as exemplified by a gemini surfactant

(a surfactant having two head groups and two hydrophobic tails joined bya short spacer).

The betaine esters of fatty alcohols are most conveniently prepared in atwo-step reaction exemplified by the following chloride-based reaction.In the first step the fatty alcohol is reacted with chloroacetylchlorideto form the chloroacetate. In a second step the chloroacetate is treatedwith trimethylamine to give the final product:R—CH₂—OH+Cl—CH₂COCl→R—CH₂—OCO—CH₂—ClR—CH₂—OCO—CH₂—Cl+N(CH₃)₃→R—CH₂—OCO—CH₂—N(CH₃)₃ ⁺Cl⁻

Both steps usually proceed in good yields and the end product, thebetaine ester, can be purified by recrystallization.

Other alcohols than fatty alcohols can be used as starting material forsynthesis of surface-active betaine esters. Non-ionic surfactants andblock copolymer surfactants, e.g. Pluronic®, which contains an alcoholgroup are also of interest for functionalisation with betaine. Alcoholsfrom natural sources are of particular interest for betaine estersurfactants to be used in life science applications. Examples of suchnatural alcohols, besides fatty alcohols, are mono- and diglycerides.Betaine esters of such alcohols can be synthesized by the proceduredescribed above but the first step, the formation of the chloroacetateintermediate, will demand higher temperature and/or longer reaction timewhen the alcohol is a secondary alcohol, as is the case for1,3-diglyceride and cholesterol, or a primary alcohol with substituentson the a-carbon, as is the case for 1,2-diglyceride.

As mentioned above, betainates of straight-chain fatty alcohols canoften be easily purified by recrystallization. When betaine esters areprepared from alcohols of natural origin, e.g. mono- or diglycerides,polymers containing hydroxyl groups or other alcohols with lesswell-defined structure, recrystallization of the product is often notpossible. In these cases purification may be accomplished by forinstance distillation or column chromatography of the intermediate orcolumn chromatography of the final product. When using starting alcoholswhere the hydroxyl group is sterically hindered, as is often the case inthe afore mentioned examples of glycerides and polymers, a quite lowyield can be expected in the first reaction step. By using a largestoichiometric excess of trimethylamine, however, a very high conversion(close to 100%) may still be achievable in the second step. In suchcases purification of the intermediate is an important part of thepreparation procedure.

An alternative way of synthesizing surface-active betaine esters from afatty alcohol is to first react betaine with chloroacetyl halogenide toform the acid halogenide, which in a subsequent step is reacted with thefatty alcohol. The reaction sequence is shown below for the chloride:C—CO—CH₂—Cl+N(CH₃)₃ ⁺→Cl—CO—CH₂—N (CH₃)₃ ⁺Cl⁻R—CH₂—OH+Cl—CO—CH₂—N (CH₃)₃ ⁺Cl⁻→R—CH₂—OCO—CH₂—N (CH₃)₃ ⁺Cl⁻

There exist yet other methods of preparing surface-active betaineesters. The present invention is not limited by the procedure used forthe synthesis of the compound.The betaine esters undergo alkaline hydrolysis much more readily thannormal esters. On the other hand, they are more resistant to acidhydrolysis than normal esters. This pronounced pH-dependence of thehydrolysis is characteristic for this class of esters and is due to thestructure of the molecules. The cationic charge in close proximity tothe carbonyl carbon of the ester bond imparts electron deficiency tothis carbon. Thus this carbon atom has a partial positive charge, whichmakes it a strong electrophile. Attack by a nucleophile at this carbonis therefore favoured compared to attack at a normal carbonyl carbon ofan ester bond. An alternative or complementary way to account for theincreased reactivity in alkaline hydrolysis of betaine ester bonds is toview the hydrolysis as a way to relief the strain of having a partialpositive charge just two atom-atom bonds away from a permanent positivecharge. The situation is shown below:

In these formulae it should be noted that R corresponds to R—CH₂— inprevious formulae.

As is also shown in the reaction scheme, acid hydrolysis is “forbidden”.It would yield a dicationic intermediate with the two positive chargesclose together and this is a very unfavourable arrangement. Takentogether, the cationic charge in the betaine ester makes alkalinehydrolysis run very fast and acid hydrolysis extremely sluggish comparedto hydrolysis of normal esters. The net effect is that betaine estersare most stable at very low pH, typically pH 2-3, and that the rate ofhydrolysis is usually pH-dependent. Already at slightly alkalineconditions, there is substantial breakdown of the molecule.

Surface-active betaine esters are even more susceptible to alkalinehydrolysis than non-surface active esters. The reason for the increasedreactivity of surface-active betaine esters is that the ester bond willhydrolyse more readily when the surfactants are in the form ofaggregates, i.e. micelles. Like all other surfactants, surface-activebetaine esters form micelles at a certain concentration, the CMC, andfurther increase of the surfactant concentration just leads to theformation of more micelles; the concentration of free surfactantmolecules stays constant. In the vast majority of applications ofcationic surfactants the concentration is far above the CMC, which meansthat almost all surfactants are in an aggregated form. The micelles ofbetaine ester surfactants are highly positively charged since all thequaternary ammonium groups are located at the surface of the aggregate.The positive charges must be compensated for by negatively charged ions,so-called counterions. All types of negative ions present in thesolution, including hydroxyl ions, will accumulate at the micellesurface. This means that the local concentration of hydroxyl ions willbe higher in the vicinity of the micelle than in the bulk, or,differently expressed, the local pH around the micelles will be higherthan in the bulk. The higher pH will cause a more rapid ester hydrolysisof micellized surfactants than of free, non-aggregated surfactantmolecules. This is referred to as micellar catalysis and is a well-knownphenomenon in physical organic chemistry. As an example, the betaineester of dodecanol at a concentration of 7.8 mM, which is 2.5 times theCMC, has a half-life of 90 minutes in a phosphate buffer of pD 7.5 at37° C. (pD is the equivalent to pH in deuterated water, D₂O).

The extent to which micellar catalysis occurs is dependent on what otheranions are present in the micellar solution. Large polarizable anions,such as bromide and iodide, will interact strongly with the micellesurface while smaller less polarizable anions, such as acetate, willhave small affinity for the micelle. This means that hydroxyl ions willcompete favourably, and be accumulated around the micelle, when thesurfactant has a small anion, such as acetate, as counterion, but thatthey will not accumulate at the micelle surface if a large ion, such asiodide, is used as counterion. Use of different counterions for thebetaine ester surfactant, and/or addition of extra salt to thesurfactant solution, is therefore a way to tune the rate of hydrolysis.

In summary, the betaine ester surfactants break down rapidly on thealkaline side and are very stable on the acid side. The hydrolysis rateis unusually pH-dependent and is also governed by the type andconcentration of anions in solution.

Cationic surfactants in general are known to interact strongly withsurfaces and many of their technical applications, such as textilesoftener, additive to fluff and tissue, hair conditioner, corrosioninhibitor, etc., rely on strong adsorption to a surface. The reason whycationic surfactants adsorb particularly strongly is that the majorityof surfaces are negatively charged, which means that a cationicsurfactant can interact with the surface by both attractiveelectrostatic forces and by hydrophobic interactions. Also biologicalsurfaces are usually negatively charged and the well documentedantimicrobial action of cationic surfactants is due to a stronginteraction with the lipid membranes of bacteria and othermicroorganisms. The strong interaction with biological lipid membranesis taken advantage of when cationic ampliphilic compounds are employedas bactericides and for gene transfection procedure mediated by cationiclipid vesicles. The strong interaction is also exploited in the use ofcationic surfactants as carriers in intracellular delivery of bioactiveagents, see U.S. Pat. No. 6,056,938. The interaction can also be aproblem in that cationic surfactants usually have a higherdermatological toxicity than other surfactants.

Betaine ester surfactants have the same adsorption characteristics asnormal cationic surfactants; thus, they adsorb strongly to negativelycharged surfaces. The driving force for adsorption increases with thelength of the hydrophobic tail of the surfactant, i.e. the R group ofthe surfactant of the formula above.

The driving force for adsorption of the betaine ester surfactants alsodepends on the ionic strength of the solution, the higher theelectrolyte concentration, the stronger the adsorption. This is a commonfeature for all ionic surfactants.

The betaine esters will form aggregates such as monolayers, bilayers orhemimicelles at surfaces, including biological surfaces. It is veryprobable, that the ester will be subject to an increased rate ofhydrolysis in such aggregates, in the same way as in aggregates insolution, i.e. micelles. Thus, “micellar catalysis” is likely to be animportant element in the determination of the life-time of adsorbedsurfactants. The betaine esters adsorbed at surfaces, as well as presentin micelles in solution, are likely to be considerably more short-livedthan free surfactant molecules in solution.

Various types of aggregates of the betaine esters, or small particles ordroplets having a surface layer of the betaine esters, will also adsorbstrongly to negatively charged surfaces. Since the cell walls arestrongly negatively charged such aggregates or particles will bindstrongly, and they will be retained at the cell surface. This isimportant for the use of these esters in drug delivery. The positivelycharged aggregates and small particles will also interact withnegatively charged polyelectrolytes, such as cell surface mucins.

Preferable embodiments of the composition according to the invention arethe following:

A water free composition which upon contact with water or otherpharmaceutical relevant aqueous medium forms collodial particles anddroplets.

A solid composition which upon contact with water or otherpharmaceutical relevant aqueous medium forms collodial particles.

A composition which upon contactwith water or other pharmaceuticalrelevant aqueous medium forms collodial particles, the pharmacologicallyactive substance being a negatively charged substance or a substancehaving low water solubility (more than 250 ml water is needed todissolve the highest dose strength).

A composition which upon contact with water or other pharmaceuticalrelevant aqueousmedium forms micelles, a microemulsion, an emulsion or adispersion of a liquid crystalline phase.

A composition which uponcontactwith water or other pharmaceuticalrelevant aqueous medium forms a dispersion of a cubic, lamellar orhexagonal liquid crystalline phase.

In this context collodial particles generally means a particle size lessthan 10 μm.

Furthermore, the composition referred to here and otherwise in thespecification means a composition containing the pharmacologically(biologically) active substance, the betaine ester and optionally otherconventional pharmaceutical excipients, such as non-ionic surface activecompounds, and/or solvents.

The compositions of the present invention can be used for improveddelivery of hydrophilic or hydrophobic pharmacologically activesubstances. The invention is not limited to the use of any specificsubstances but preferably do the biologically active substances have lowwater solubility or are negatively charged substances. Examples ofbiologically active substances include, but are not limited to, nucleicacids such as DNA, cDNA, RNA (full length mRNA, ribozymes, antisenseRNA), oligodeoxynucleotides (phosphodiesters, phosphothioates,phosphoramidites, and all other chemical modifications), oligonucleotide(phosphodiesters, etc.) or linear and closed circular plasmid DNA;negatively charged proteins and carbohydrates including polysaccharides.Suitable drugs include antivirals (acyclovir, IUdR, ganciclovir,vidarabine, AZT), steroidal and non-steroidal anti-inflammatory drugs(dexamethasone, loteprednol, prednisolone derivatives, diclofenac,indomethacin, piroxicam etc.), antibiotics (e.g., ampicillin anderythromycin) antifungals (e.g., miconazole), vitamins, hormones,retinoic acid, local anesthetics, calcium channel blockers (e.g.,Verapamil), prostaglandins and prostacyclins, antineoplastic andantimetabolitic drugs, miotics, cholinergics, adrenergic antagonists,anticonvulsants (e.g., phenytoin), antianxiety agents, majortranquilizers, antidepressants, anabolic steroids, estrogens,progesterones, and glycosaminoglycans (heparin, heparan, chondroitinsulfate, and low molecular weight derivatives thereof).

“Ionic interaction” or “electrostatic interaction” refers tointermolecular interaction among two or more molecules, each of which ispositively or negatively charged. Thus, for example, can positivelycharged lipids interact with negatively charged molecules like DNA. Genetransfer represents an important advance in the treatment of bothgenetic and acquired diseases. Cationic lipid-mediated gene transferhave advantages over viral gene transfer due to their non-immunogenicproperties.

Many of the drugs listed above have low bioavailably when administeredorally due to low water solubility, slowly transport through mucous, lowpermeability through the epithelial cells, instability in biologicalfluids or a combination of these factors. Low bioavailability of suchdrugs severely limits their applicability, usage and effectiveness.

Typical pharmaceutical applications of the invention are in oraladministration or transmucosal delivery of sparingly water solubledrugs. For oral administration a composition containing the drug and thebetaine ester is encapsulated in a sealed soft or hard gelatin capsule.The capsule is typically of a kind which is dissolved in a particularregion of the GI tract where it releases its content. Examples of suchcapsules are entero-coated soft or hard gelatin capsules. Entericcoating, as known per se, is a coating consisting of a substance or acombination of substances that resists dissolution in gastric fluid butdisintegrates in the intestine. The formation of well-defined colloiddrug containing particles and droplets (e.g. liposomes, microemulsions,emulsions, Cubosome® or Hexosome® particles) when the capsule isdisintegrated brings about predictable release of the drug which mayoffer an improvement in both the rate and extent of absorption. Estersof long chained fatty acids in lipid drug delivery dispersions willafter digestion and absorption be transported via the lymphatic systemin so called chylomicrons. The chylomicrons are in turn carried awayfrom the small intestine through the thoracic duct, thus bypassing theliver. Such an absorption route thus significantly reduces the firstpass effect of drug absorbed together with the lipids.

Dosage forms of the compositions of pharmacologically active substances,betaine esters and any other excipients can be fluid, semisolid orsolid. Betaine esters and biologically active substances my be combinedwith other excipients so that they are fluid at elevated temperaturewhich allows for filling capsules followed by formation of a solidsolution, a solid dispersion or a semisolid formulation when thecapsules are stored at room temperature. The term semisolid should beinterpreted in the common way in this technical field, i.e. generally aformulation that does not flow under its own weight. Normally this alsomeans that it is semisolid at room or ambient temperature and can beliquefied at higher temperatures.

Inclusion of surfactants in lipid-based liquid crystalline drug deliveryparticles or precursor systems of such particles can improve loading ofwater soluble drugs. The enhanced loading of the negatively chargedwater-soluble drug ketoprofen by the inclusion of cationic surfactantsinto Cubosome® particles have been demonstrated in the literature.Development of lipid-based particles like Cubosome®, Hexosome® andliposomes containing the betaine esters and water soluble drugs is anapplication of the invention. Another interesting application of theinvention is formation of positively charged aggregates of a dispersedlamellar liquid crystalline phase containing the betaine ester and drugthat with time undergoes phase transition due to hydrolysis of thebetain ester, thereby altering the drug delivery properties of theparticles.

In one embodiment of the invention the betaine ester is combined withother lipid excipients like PC in order to lower the toxicity of thedrug delivery vehicle. The reduction in toxicity may be evaluated byhaemolysis experiments.

DNA transfection efficiency of aggregates containing cationic lipids canbe modified by co-formulating with neutral “helper lipids” likedioleoylphosphatidyl-ethanolamine (DOPE), cholesterol or poly(ethyleneglycol)-phospholipid conjugate. Of special interest is co-formulation ofthe betaine esters with polar lipids that promote formation ofnon-lamellar structures, e.g. phosphatidylethanolamine (PE).

Cryoprotectants and polymers are examples of components that mayoptionally be used in the drug delivery systems based on betaine esters.A cryoprotectant or anticoalescent compound may be added to aformulation of betaine ester and drug prior to dehydration/evaporationto inhibit flocculation and coalescence upon rehydration. Thecryoprotectant may be of any type known in the art, including sugars andpolysaccharides such as sucrose or trehalose, and nonnatural polymerssuch as polyvinylpyrrolidone. Cryoprotectants are usually present atless than 25%, commonly 10%, more commonly 5%, 4% (w/v) or less in theemulsion before lyophilization. Natural polymers, synthetically modifiednatural polymers, such as (hydroxypropyl) methylcellulose or syntheticpolymers, such as polyvinylalcohol may also be included in betaine esterformulations in order to modify the release of the drug carryingaggregate/particle

The betaine ester-containing composition according to the invention maybe prepared by use of water or other solvents followed by evaporation,wherein the evaporation is accomplished by spray drying, freeze drying,air drying, vacuum drying, fluidized bed drying, co-precipitation, orsuper-critical fluid evaporation.

A further aspect of the invention provides a dehydrated colloidalsuspension. Dehydrated suspensions may be stored for prolonged periodswith minimal degradation, and can be reconstituted with water shortlybefore use. The residual water content in an dehydrated emulsion isusually less than 5% (w/w), commonly less than 2%, and often less than1%.

Dehydration may be performed by standard methods, such as drying underreduced pressure; when the suspension is frozen prior to dehydration,the low pressure evaporation is known as lyophilization. Freezing may beperformed in a dry ice-acetone or ethyl alcohol bath. The pressurereduction may be achieved with a mechanical vacuum pump, usually fittedwith a liquid nitrogen cold trap to protect the pump from contamination.Pressures in the low millitorr range, e.g., 10-50 millitorr, areroutinely achievable, but higher or lower pressures are sufficient.

Especially preferable embodiments of the invention are compositions inthe form of freeze-dried powder, spry-dried powder and a pumpable massthat can be filled into a capsule.

The pharmacologically effective amount of the active substance is ofcourse chosen, by the person skilled in the art, along known principles,while taking into consideration which specific compound is selected, thespecific use therof and so on. Similarly the concentrations ofexcipients, solvents, etc. are also selected in accordance with priorart so as to achieve the desired solid, semisolid or fluid state.Finally, the percentage of the betain ester is also easily determined bythe skilled artisan while considering known principles concerningcationic excipients and the specific purposes to be obtained.

EXAMPLES Example 1

Synthesis of dodecyl betainate

Dodecyl betainate was prepared from dodecanol, choloroacetyl chlorideand trimethylamine using the two-step synthetic procedure describedbelow:

In the first step chloroacetyl chloride (14.2 g, 126 mmol) indichloromethane (25 ml) was drop-wise added to a stirred solution of1-dodecanol (22.73 g, 122 mmol) in dichloromethane (100 ml). Thereaction mixture was stirred for 6 h at room temperature and then gentlyrefluxed for 0.5 h. After being washed with a 5% solution of sodiumhydrogen carbonate (3×25 ml), to remove excess chloroacetyl chloride,the organic phase was dried over magnesium sulphate, filtered and rotaryevaporated.

In the second step trimethylamine (14.0 g, 237 mmol) was slowly bubbledthrough an ice-cooled, stirred solution of dodecyl chloroacetate (15.24g, 58 mmol) in dry acetone (600 ml), whereupon the solution was allowedto attain room temperature. After 76 h the product, a white fluffyprecipitate, was collected on a glass filter and washed with diethylether (3×20 ml). The product weighed 16.2 g, corresponding to an overallyield of 83%. ¹H-NMR (CDCl₃): δ 0.88 (t, 3H), 1.22-1.39 (m, 18H), 1.66(m, 2H), 3.66 (s, 9H), 4.18 (t, 2H), 5.19 (s, 2H)

Example 2

Concentration Dependence on the Hydrolysis of Surface Active betaineesters

FIG. 1 shows the concentration dependency of the hydrolysis rate forsurface active betaine esters, exemplified by the initial pseudofirst-order rate constants versus concentration for a number of betaineesters ((∘) Oleyl betainate, (●) tetradecyl betainate, (□) dodecylbetainate, (▪) decyl betainate, (⋄) ethyl betainate) in a phosphatebuffer of pD=7.5 at 37° C. (pD is the equivalent to pH in deuteratedwater, D₂O). Ethyl betainate is included as a non surface activereference. The increase in hydrolysis rate with increasing concentrationis caused by an increasing contribution from micellar catalysis, and thefollowing decrease can be explained by the increased competition betweenthe hydroxyl ions and the surfactant counterions at the micellarsurface.

FIG. 1 also shows the dramatic increase in hydrolysis rate due to thepresence of micellar catalysis for the surface active betaine esters.For instance, dodecyl betainate at a concentration of 7.8 mM, which is2.5 times the CMC, has a half-life of 90 minutes, compared to ahalf-life of 9 h for ethyl betainate.

Example 3

Using Chamber Experiments to Show Low Toxicity of betainate Systems

A modified Using diffusion chamber with an exposed tissue area 1.78 cm²was used in the experiments. 15 to 20 cm of the small intestine, distalto the Ligament of Treitz was removed from rats (male Sprague-Dawley,400-500 g) and used in the tests. Three rats and three segments per ratwere included in each experimental group. The passage of differentmarker molecules, ¹⁴C-mannitol and FITC-dextran (4400 FD4), from themucosal to the serosal chamber were expressed as apparent permeabilitycoefficient (Papp). The apparent permeability coefficient (Papp) tomannitol and FITC-dextran 4.400 across the intestinal mucosa wascalculated from the equation: Papp (cm/s)×10-6)=dc/dt * (V/(A*C0)),where dc/dt is the change of the serosal concentration over time(mol/L/sec), V is the volume in the reservoir of the serosal side (cm³),CO is the initial concentration of the marker in the mucosal reservoir(mol/L), and A is the exposed intestinal area in the chamber (cm²) . Theexperiments were done on marker molecules dissolved in a dodecylbetainate (0.5 % w/w) containing solution and in a controlsolution(Krebs buffer).

The Papp values for 14C-mannitol are scattered between 3 and 4×10⁻⁶cm/s. There are no obvious differences between the different treatments.

The Papp values for FD4 are scattered between 0.4 and 0.6×10⁻⁶ cm/s(App. 2). There are no obvious differences between the differenttreatments.

The difference in electric potential over the segments was measuredbefore and after the experiment. With respect to permeability for markermolecules, the intestinal segments were not affected by co-formulationwith dodecyl betainate. Furthermore there was no increased damage tosegments in response to treatment with betaine ester containing solutionas compared to control, assessed by determination of potentialdifference over the segments before and after treatment.

Example 4

Hemolysis Experiments on betaine ester Surfactants

Erythrocytes from rats were washed 3 times and suspended in salinesolution (0.9%) to a volume fraction of 0.025. The hemolys experimentswere performed by mixing the erythrocyte suspension with surfactantsolutions in a 1:1 ratio, thereby giving a finale erythrocyte volumefraction of 0.0125 and a surfactant concentration as stated in thefigure. The samples were then incubated at 37° C. for 1 hour andcentrifuged at 2000 g for 10 minutes. Aliquots of the supernatant wasmixed with an equal volume of 15 mM C14TAC before the hemoglobin contentwas measured spectrophotometrically on a Lab systems iEMS Reader MF at540 nm.

For the samples containing phospholipids, a liquid concentrate ofdodecyl betainate, PC and ethanol in a weight ratio of 16:64:20 wereprepared. The concentrate was dispersed in saline solution by gentlestirring at room temperature just before the experiment. The resultingaqueous dispersion was tested in the same way as stated above for thesurfactant solution.

FIG. 2 shows the relative haemolytic effect of different cationicsurfactant solultions and a cationic ionic phospolipid dispersion. Theconcentrations given in the figure represent the amounts of cationicsurfactant in the tested solutions. OB=oleyl betainate, DDB dodecylbetainate, DDB/PC=dodecyl betainate coformulated with phosphatidylcholine in a weight ratio of 20 to 80 (concentration of DDB given),C14TAC=tetradecyltrimethylammonium chloride. The phosphatidyl cholineused is Epikuron 200 from Degussa BioActives, a pharmaceutical gradeproduct of soybean origin. All data are normalized to the absorbanceobtained from hemolysis by the 0.5 mM tetradecyltrimethylammoniumchloride sample.

This example shows that at low concentrations the toxicity to red bloodcells of dodecyl betainate is lower than that of the stable surfactantC14TAC, which has a comparable critical micelle concentration 2.5 mM(CMC for dodecylbetainate is 3.1 mM) and that the toxicity of can befurther lowered by co-formulating the betaine ester with phosphatidylcholine.

Example 5

Aqueous Particle Dispersion of a Sparingly Water Soluble SubstanceStabilized With betaine esters.

The ability of dodecyl betainate to stabilize aqueous dispersions ofhydrophobic particles was investigated in samples containing 1%cholesterol (of lanolin origin, Fluka) in pure water and in dodecylbetainate solutions of varying concentration. The samples were preparedby weighing the components into screw-capped glass vials that were firstshaken, then kept in an ultrasonic bath for 5 minutes and finally shakenagain before left for inspection. The table below presents the visualappearance of samples with different surfactant concentrations atdifferent times. Dodecyl Visual appearance Visual appearance betainateconc. after 5 min. after 60 min. 0 Coarse dispersion of Almost clearwater flake-like particles and precipitate 0.1% w/w (3 mM) Turbiddispersion Very slightly turbid dispersion and precipitate 0.3% w/w (9mM) Highly turbid Highly turbid dispersion dispersion and someprecipitate

The sample with pure water was a coarse dispersion that precipitatedquickly, while samples containing more than approximately 0.1% of thesurfactant were fine dispersions with prolonged stability. The tableshows that particles of water insoluble particles like steroids caneasily be stabilized against particle aggregation and sedimentation bybetainate esters in aqueous systems.

Example 6

Transformation From a Lamellar Liquid Crystalline Phase to a Cubic PhaseWith time in Mixture of dodecyl betainate, Glycerol Monooleate and aPhysiologically Relevant Medium

A solution A was made from 81% w/w glycerol monooleate (DaniscoBrabrant, Danmark), 9% w/w dodecyl betainate and 10% w/w ethanol. Twosamples were prepared by mixing solution A with simulated intestinalfluid (SIF) (pH=7.4) and with water, respectively. The compositions ofthe samples are reported in the table below. The phase behaviour of thesamples as a function of time was studied by visual inspection betweencrossed polarizers and reported in the table below. A sample of glycerolmonooleate and water was used as a reference. Glycerol monooleate isknown to form a cubic, nonbirefingent liquid crystalline phase in excesswater at room temperature. Composition Time 0 h Time 0.5 h Time 14 h0.25 g A/1.8 g Dispersion Homogenous Homogenous, water birefingentbirefingent 0.27 g A/1.8 g Dispersion Homogenous, Two non SIFbirefingent birefingent phases Glycerol — — Two non monooleate 0.3 g/birefingent water 1.8. phases

This experiment shows that addition of dodecyl betainate to glycerolmonooleate induces a phase shift from a cubic phase to a birefingentphase, probably a lamellar phase and that this lamellar phase atphysiological relevant conditions with time transforms into two phases,probably cubic liquid crystalline phase and an aqueous solution.

1. A pharmaceutical composition comprising a pharmaceutically effectiveamount of a pharmacologically active substance and a pharmaceuticallyacceptable carrier comprising a cationic excipient, wherein saidcationic excipient is a labile ester of betaine and a lipophilic alcoholhaving at least one primary hydroxyl group.
 2. A composition accordingto claim 1, wherein said carrier is substantially non-aqueous.
 3. Acomposition according to claim 1, wherein said composition issubstantially non-aqueous.
 4. A composition according to claim 1,wherein said lipophilic alcohol is an alcohol of the formula R—CH₂—OHwhere R is a saturated or unsaturated aliphatic hydrocarbon residuehaving 7-30 carbon atoms.
 5. A composition according to claim 4, whereinsaid hydrocarbon residue has at least one primary and/or at least onesecondary hydroxyl groups as substituent(s).
 6. A composition accordingto claim 5, wherein said primary and/or secondary hydroxyl groups areeach at most two.
 7. A composition according to claim 4, wherein saidhydrocarbon residue is interrupted by at least one oxygen atom.
 8. Acomposition according to claim 7, wherein said oxygen atom(s) is (are)present in an ester linkage.
 9. A composition according to claim 8,wherein said ester linkage is present in a glyceride.
 10. A compositionaccording to claim 9, wherein said glyceride is a 1-monoglyceride or a2-monoglyceride.
 11. A composition according to claim 4, wherein saidunsaturated hydrocarbon residue is a residue containing one or twodouble bonds.
 12. A composition according to claim 1, wherein saidpharmacologically active substance has low water solubility, such ascyclosporine or an analogue thereof.
 13. A composition according toclaim 1, wherein said pharmacologically active substance is negativelycharged.
 14. A composition according to claim 1, which has a watercontent of at most 5% by weight.
 15. A composition according to claim 1,which is solid or semi-solid.
 16. A composition according to claim 15,which is in the form of a powder or a waxy powder.
 17. A compositionaccording to claim 15, which is a pumpable mass that can be filled intoa capsule.
 18. A composition according to claim 1, which upon contactwith water or other aqueous medium forms colloidal particles.
 19. Acomposition according to claim 18, wherein said pharmacologically activesubstance is a negatively charged substance or a substance having a lowwater solubility.
 20. A composition according to claim 1, which uponcontact with water or other aqueous medium forms micelles, amicroemulsion, an emulsion or a dispersion of a liquid crystallinephase.
 21. A composition according to claim 1, which upon contact withwater or other aqueous medium forms a dispersion of a cubic, lamellar orhexagonal liquid crystalline phase.
 22. A composition according to claim1, wherein said carrier also comprises an excipient selected frompolymers, lipids, carbohydrates, non-ionic surface active compounds andmixtures thereof.
 23. A composition according to claim 22, wherein saidcarbohydrate is a low molecular carbohydrate.
 24. A compositionaccording to claim 22, wherein said lipid is selected fromphospholipids, cholesterol and glycerides from medium- or long-chainedfatty acids. 25-29. (canceled)
 30. A composition according to claim 2,wherein said composition is substantially non-aqueous.
 31. A compositionaccording to claim 1 wherein said pharmacologically active substance isnegatively charged and is selected from the group consisting ofcarbohydrates, low molecular weight derivatives of heparin, and DNA. 32.A composition according to claim 1, which has a water content of at most2% by weight.
 33. A composition according to claim 1, which has a watercontent of at most 1 % by weight.
 34. A composition according to claim1, wherein said carrier also includes a carbohydrate selected from thegroup consisting of lactose, sucrose, maltose, and trehalose.
 35. Amethod for administering a pharmaceutically effective amount of apharmacologically active substance with a cationic excipient including alabile ester wherein hydrolysis does not occur until administered,comprising administering the pharmaceutical composition of claim
 3. 36.A method for administering a pharmaceutically effective amount of apharmacologically active substance with a cationic excipient including alabile ester, comprising administering the pharmaceutical composition ofclaim 1.